Effect of guanidinoethanesulfonic acid on brain monoamines in the mouse

Guanidinoethanesulfonic acid (GES) is known to induce convulsive seizures when administered intracisternally to rabbits and cats. The effects of GES on behavior, electroencephalographic recording and brain monoamine levels were examined in mice. When GES (900 nmol) was intraventricularly injected into mice, focal clonic movements of the face, vibrissae and ears together with twitching of the limbs were observed 0.5–1 min after the injection. Hypersensitivity was observed up to 7 min after the injection, after which the mice behaved normally. GES also induced sporadic spike discharges on electrocorticogram. The levels of 5-hydroxytryptamine (5-HT) of the GES-injected mice were lower than those of the saline-injected mice in the hippocampus, diencephalon, pons-medulla oblongata and cerebellum 5 min after the injection. No changes in the norepinephrine or dopamine levels were found after the GES injection. The level of 5-hydroxyindoleacetic acid increased in the striatum and cerebellum 5 min after the GES injection. These results suggest that GES-induced convulsive activities enhance the serotonergic neuroactivity in order to suppress the convulsions.     

Collagen-gel-embedded three-dimensional culture of human thyroid epithelial cells: comparison between the floating sandwich method and the dispersed embedding method.

Human thyroid epithelial cells were isolated from surgically resected human thyroid gland with collagenase and cultured for one week under EGF-supplemented conditions to allow them to proliferate. Then the cells were transferred to the following three-dimensional culture systems. One was a culture of isolated cells between floating double layers of collagen gel, designated the "floating sandwich method." The other was a culture of isolated cells mixed with collagen gel, designated the "dispersed embedding method." Many folliclelike structures with lumina of appreciable size were obtained by the former method. The cells cultured by the floating sandwich method exhibited a distinct polarity shown by the presence of numerous microvilli at the apical surface and close contact with collagen gels at the basal surface. On the other hand, only a few folliclelike structures were obtained by the dispersed embedding method, in which the folliclelike structures were small in size and the cells showed less distinct polarity than those observed in the floating sandwich method. Thus, the floating sandwich method appears to be suitable for studying the process and mechanism of in vitro organization of follicular structures by human thyroid epithelial cells.     

Human recombinant interleukin-1 alpha-mediated stimulation of procollagenase production and suppression of biosynthesis of tissue inhibitor of metalloproteinases in rabbit uterine cervical fibroblasts

Influence of human recombinant interleukin-1 (hrIL-1) on collagen metabolism was investigated with rabbit uterine cervical fibroblasts. Enzyme-linked immunosorbent assays for collagenase and tissue inhibitor of metalloproteinases (TIMP) indicated that hrIL-1 participates in both stimulation of procollagenase production and suppression of TIMP synthesis by uterine cervical cells. IL-1 did not modulate collagen synthesis. In addition, the sensitivity to IL-1 of uterine cervix from ovariectomized rabbits was augmented by estradiol-17 beta treatment. Thus it is proposed that IL-1 accelerates collagenolysis in the cervical tissue and its effect on uterine cervix is hormonally regulated.     

Effects of monoenergetic X-rays with resonance energy of bromine K-absorption edge on bromouracil-labelled E. coli cells

In order to examine enhanced killing that might be induced by Auger cascades in the incorporated atoms in cells, bromouracil(BrU)-labelled E. coli cells were irradiated with monoenergetic X-rays at 13.49 and 12.40keV, just above and below the K-absorption edge of bromine. In both cases BrU-labelled cells were more sensitive for killing than were normal cells. However, when the degree of BrU-sensitization was compared between the two energies of X-rays, the enhanced killing at 13.49 keV was only small, 2 +/- 8 per cent based on the D0 value in saline. By the addition of DMSO, which is believed to suppress radical-mediated effects, killing of BrU-labelled cells was enhanced at 13.49 keV by 8 +/- 4 per cent as compared with 12.40 keV, based on D0. These results have been examined in terms of absorbed energy in BrU-labelled cells and in terms of the number of induced Auger events.     

Chromosomal genes essential for stable maintenance of the mini-F plasmid in Escherichia coli

We have isolated mutants of Escherichia coli which do not support stable maintenance of mini-F plasmids (delta ccd rep+ sop+). These host mutations, named hop, were classified into five linkage groups on the E. coli chromosome. Genetic analyses of these hop mutations by Hfr mating and P1 transduction showed their loci on the E. coli genetic map to be as follows: hopA in the gyrB-tnaA region, hopB in the bglB-oriC region, hopD between 8 and 15 min, and hopE in the argA-thyA region. Kinetics of stability of the sop+ and delta sop mini-F plasmids in these hop mutants suggest that the hopA mutants are defective in partitioning of mini-F rather than in plasmid replication. The hopB, hopC, and hopD mutants were partially defective in replication of mini-F. The physical structure of the plasmid DNA was normal in hopA, B, C, and D mutants. Large amounts of linear multimers of plasmid DNA accumulated in mutants of the fifth linkage group (hopE). None of the hop mutations in any linkage group affected the normal growth of cells.     

Glycosaminoglycan polysulfate-induced stimulation of hyaluronic acid synthesis in rabbit knee synovial membrane: involvement of binding protein and calcium ion

We have previously reported that naturally occurring sulfated glycosaminoglycans having a chondroitin-type structure and glycosaminoglycan polysulfate (GAGPS, a persulfated derivative of chondroitin sulfate) caused a specific stimulation of hyaluronic acid synthesis in rabbit knee synovial membranes [H. Nishikawa et al. (1985) Arch. Biochem. Biophys. 240, 146-153]. In the present study, the interaction of [3H]GAGPS and the surface of the rabbit knee synovial membranes and the relationship between this interaction and the stimulatory effect of GAGPS on the hyaluronic acid synthesis were examined in order to define the stimulatory mechanism of hyaluronic acid synthesis by GAGPS. A part of the [3H]GAGPS taken up by the synovial membranes was released from the membranes by trypsin treatment. The interaction of [3H]GAGPS and the surface of the isolated synovial membranes was diminished by pretreatment of the membranes with proteases or chelating reagents. Pretreatment of synovial membranes with trypsin or ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid had little effect on the basal hyaluronic acid synthesis but caused the loss of GAGPS-induced stimulation of hyaluronic acid synthesis accompanied by significant decrease (20% P less than 0.05-P less than 0.01) in the interaction between GAGPS and the surface of the synovial membranes. Dermatan sulfate having a chondroitin-type structure also stimulated hyaluronic acid synthesis but this effect was not additive to the stimulation of hyaluronic acid synthesis by GAGPS. Heparin had no effect on either the basal hyaluronic acid synthesis or the GAGPS-induced stimulation of hyaluronic acid synthesis. These results indicate that binding of GAGPS to certain distinct protein components on the surface of synovial membranes is involved in the stimulatory mechanism of hyaluronic acid synthesis by GAGPS, and that the binding may be mediated by Ca2+ ion. The binding was also found to be specific for sulfated glycosaminoglycans having a chondroitin-type structure.     

Primary structure of non-histone protein HMG1 revealed by the nucleotide sequence

The isolation and sequencing of a cDNA clone coding for the entire sequence of pig thymus non-histone protein HMG1 are described. The sequence analysis reveals a complete 2192-nucleotide sequence with a 5'-terminal untranslated region of 11 nucleotides, 642 nucleotides of an open reading frame that encoded 214 amino acids, and a 3'-terminal untranslated region of 1539 nucleotides. The HMG1 protein, deduced from the nucleotide sequence, has a molecular weight of 24,785 and a C-terminal of a continuous run of 30 acidic amino acids, encoded by a simple repeating sequence of (GAN)30. The predicted amino acid sequence is homologous to HMG1, HMG2, and HMG-T sequences from several sources, suggesting that the protein conformation is under evolutionary constraints. Northern blot analysis reveals that another hybridizable RNA species of smaller size is present. Southern blot analyses suggest that pig genome contains several HMG1 gene equivalents.     

Increase in peripheral large granular lymphocytes in postpartum autoimmune thyroiditis

In order to elucidate the mechanism of postpartum aggravation of autoimmune thyroid disease (AITD), we serially examined the change in the proportion of peripheral large granular lymphocytes (LGL), which have activities of NK, K and/or cytotoxic T cells, in their postpartum period. Within 6 months postpartum, the percentage of LGL increased transiently in patients with AITD who remained euthyroid, or developed destructive thyrotoxicosis and/or hypothyroidism due to thyroiditis and even in normal controls. These changes in the LGL percentage were more obvious in the patients who had marked postpartum thyroid dysfunction. In contrast, we did not find a definite increase in the LGL percentage within 6 months postpartum in patients with Graves' disease who relapsed into Graves' thyrotoxicosis. These deta suggest that the increase in LGL in the postpartum period may be related to the induction of postpartum destructive thyrotoxicosis and/or hypothyroidism in AITD.     

A noncleavable signal for mitochondrial import of 3-oxoacyl-CoA thiolase

Rat 3-oxoacyl-CoA thiolase, an enzyme of the fatty acid beta-oxidation cycle, is located in the mitochondrial matrix. Unlike most mitochondrial matrix proteins, the thiolase is synthesized with no transient presequence and possesses information for mitochondrial targeting and import in the mature protein of 397 amino acid residues. cDNA sequences encoding various portions of the thiolase were fused in frame to the cDNA encoding the mature portion of rat ornithine transcarbamylase (lacking its own presequence). The fusion genes were transfected into COS cells, and subcellular localization of the fusion proteins was analyzed by cell fractionation with digitonin. When the mature portion of ornithine transcarbamylase was expressed, it was recovered in the soluble fraction. On the other hand, the fusion proteins containing the NH2-terminal 392, 161, or 61 amino acid residues of the thiolase were recovered in the particulate fraction, whereas the fusion protein containing the COOH-terminal 331 residues (residues 62-392) was recovered in the soluble fraction. Enzyme immunocytochemical and immunoelectron microscopic analyses using an anti-ornithine transcarbamylase antibody showed mitochondrial localization of the fusion proteins containing the NH2-terminal portions of the thiolase. These results indicate that the NH2-terminal 61 amino acids of rat 3-oxoacyl-CoA thiolase function as a noncleavable signal for mitochondrial targeting and import of this enzyme protein. Pulse-chase experiments showed that the ornithine transcarbamylase precursor and the thiolase traveled from the cytosol to the mitochondria with half-lives of less than 5 min, whereas the three fusion proteins traveled with half-lives of 10-15 min. Interestingly, in the cells expressing the fusion proteins, the mitochondria showed abnormal shapes and were filled with immunogold-positive crystalloid structures.